Optical microscopy, and particularly fluorescence microscopy, is a powerful tool in biomedicine and can be applied with exogenous fluorescence labels to study, for example, the distribution of biological components (cells, extracellular matrix material, proteins, metabolites etc.) and their interactions. Biological cells and tissue are themselves fluorescent and this “autofluorescence” signal can also be used to learn about cellular and tissue samples to better understand disease, to study the effect of potential therapeutic agents, and to diagnose disease. Increasingly there is a drive to study molecular biology in vivo in living organisms (e.g. animals) and to gain more information from autofluorescence signals in humans for diagnostic and other applications. Fluorescence microscopes can not only provide intensity-based imaging but can also resolve the fluorescence spectrum and lifetime and polarisation properties. Confocal microscopy provides improved contrast, spatial resolution and optical sectioning compared to wide-field microscopes. Multiphoton microscopes utilise the nonlinear scaling of the excitation process to realise optical sectioning and can confer advantages of reduced photobleaching and reduced attenuation due to absorption and scattering in biological tissue compared to wide-field or confocal microscopes. Unfortunately, the strong optical scattering associated with biological tissue limits the imaging depth of conventional microscopes to typically a few 100 μm. Multiphoton microscopes can image to deeper depths since the longer wavelength excitation radiation experiences reduced attenuation in biological tissue but the imaging depth is still limited to <<1 mm. For these reasons, there is significant interest in using endoscopes to image deeper in biological samples, including animals and humans.
Current endoscopes may be considered in the categories of flexible video endoscopes, rigid optical endoscopes and flexible optical endoscopes. Video endoscopes typically have a miniature CCD camera at the distal end and the flexible section of the endoscope is essentially a cable conduit for the electronic signals and power etc. Video endoscopes are essentially wide-field imaging instruments with an optical performance that can be considered as a wide-field microscope. Rigid optical endoscopes are typically constructed from a series of lenses enclosed in a rigid cylinder and these relay an optical image from the distal to proximal end. They are usually employed as wide-field microscopes with a CCD camera at the proximal end but they can be used in scanning microscope configurations [1] and this approach has recently found favour in multiphoton microscopy where a “stick lens” made from gradient-index (“GRIN”) lenses is employed.
Rigid endoscopes are typically used in orthopaedic surgery or surgery in the large body cavities, and for imaging in rodent brains. They are not usually suitable to study internal organs because they are not flexible enough to be passed through internal pathways, small body cavities or vessels in live subjects, and they are typically of limited length. For internal imaging, it is usual to employ flexible endoscopes. For intensity imaging, video endoscopes are most commonly used but for more sophisticated imaging modalities such as hyperspectral imaging or fluorescence lifetime imaging or confocal or multiphoton microscopy (to provide higher resolution and optical sectioning), it is necessary to use a flexible optical endoscope. Flexible optical endoscopes can be divided into wide-field optical endoscopes and microconfocal endoscopes and multiphoton endoscopes.
Wide-field (non-confocal) flexible optical endoscopes typically utilise a fibre bundle to relay the optical image from the sample (distal) end to the detector (proximal) end, as illustrated in FIG. 1(a). These fibre bundles typically comprise ˜30,000 optical fibres that each correspond to an image pixel, with the fibre bundle being about 0.6 mm in diameter. This is a small number of pixels compared to a typical CCD camera and so such optical endoscopes offer a smaller number of image resolution elements than video endoscopes or optical microscopes and consequently lower quality images. Cross-talk arising from leakage of light between different optical fibres in the fibre bundle can also degrade the image. The spacing between individual fibre cores (and consequent fill-factor) also impacts the efficiency of light collection and the image quality.
Microconfocal endoscopes either utilise a proximal scanner with an imaging fibre bundle (FIG. 1(b)) or a distal scanner with a single optical fibre (FIG. 1(c)) to convey the light from the sample to the (proximal) detector. In the former case, the fibre bundle can be an array of single mode fibre “cores” that are fabricated together to form a “coherent” bundle.
For the fibre bundle-based microconfocal endoscope [2], the scanner at the proximal end (FIG. 1(b)) scans the excitation beam across the proximal end of the fibre bundle, addressing each optical fibre core sequentially, and the output at the distal end is relayed by the objective lens to scan a focussed beam across the sample. The resulting fluorescence (or reflected light) is imaged back to the same fibre core and the image of the sample is thus relayed to the proximal end of the fibre bundle. This can be imaged directly, e.g. using a CCD, or propagated back through the scanning system to a single detector that records the pixel information sequentially. As with the wide-field endoscope, the limited number of fibre cores in the imaging bundle limits the image quality. When imaging in scattering media such as biological tissue, there can be cross-talk arising from light collected by other fibre cores than the one addressed by the scanner. There can also be leakage of light between the single mode fibres that can contribute to cross-talk. Each single mode fibre core acts as a “confocal pinhole” (and an additional confocal pinhole may also be deployed in front of the detector), leading to optical sectioning and improved resolution and contrast compared to wide-field imaging. Focus adjustment or axial (depth scanning) may be realised by translating the objective lens assembly relative to the distal end of the fibre.
The single mode optical fibre microconfocal endoscope represented in FIG. 1(c) employs a scanner at the distal end, e.g. [3]. The excitation light emerges from the distal end of the single mode fibre, which here serves as the “confocal pinhole”. Given the nature and applications of endoscopy, the components at the distal end are required to be small, and so considerable effort has been invested in developing miniature optical scanners. These can be based on microfabricated scanning mirrors or on vibrating fibre tip designs. To date miniaturisation has permitted endoscopes with diameters of a few mm to be developed, but this is still too large for some desired purposes. In general, the fibre-bundle microconfocal endoscope, which does not require a distal (x-y) scanner, can be made thinner than the single mode fibre/distal scanner approach, making it potentially more flexible and able to pass through thinner cavities or vessels etc.
Both the fibre bundle and single fibre approaches to microconfocal endoscopy can be adapted to multiphoton imaging, which can offer deeper penetration in biological tissue. Unfortunately, since the single mode fibre (or fibre core in the bundle) acts as the confocal pinhole, this removes one of the advantages of multiphoton endoscopy—namely that the confocal pinhole is not needed (since all multiphoton excited photons should originate from the focus) and in scattering media an open pinhole permits more signal to be collected. One way round this for the fibre bundle approach would be to use a large area detector in place of the CCD camera indicated in FIG. 1(b) although the single mode fibre cores would still act as apertures and not collect all of the multiphoton excited fluorescence.
To summarise, microconfocal (and multiphoton) endoscopes offer significant advantages over wide-field endoscopes (video or flexible optical fibre bundle) including optical sectioning (and therefore subsurface imaging), superior image contrast (S/N) and improved lateral resolution. The fibre bundle approach suffers from reduced image quality due to the limited number of pixels, which is a consequence of the number of single mode fibre cores in available fibre bundles and the spacing between the fibre cores. This results in undersampling of the image and the limited fill-factor also impacts light collection efficiency. Cross-talk between different fibre cores—and that arising from any light entering the bundle between the fibre cores—can also be an issue. The single fibre/distal scanner approach can provide a high resolution (fully sampled) image but the size of the scanner means that it is difficult to make a very thin endoscope with this approach. For both approaches a z-position/focussing adjustment usually requires moving parts at the distal end. Multiphoton endoscopy is usually implemented via the single fibre/distal scanner approach, for which the restriction to the fixed pinhole of the optical fibre is a drawback when imaging in scattering media, although this drawback can be mitigated by the use of specially designed fibres such as conventional or microstructured double clad fibres.
There is therefore a desire to reduce the components used at the distal end of the endoscope. In particular, it would be desirable to achieve the (fully sampled image) performance of the distal scanner approach, but without the need for a distal scanner, thereby permitting thinner and more compact endoscopes. For the same reasons, it would also be beneficial to be able to manage without an objective lens at the distal end of the endoscope. It would also be desirable to be able to use fewer fibres in the fibre bundle, in order to be able to reduce the diameter of the fibre bundle and increase its flexibility.